This invention relates to reagents and methods for rapidly and quantitatively assaying the concentration of analytes in biological samples. More particularly, this invention provides assays having a wide dynamic working range by simultaneously incubating a sample which may contain the analyte(s) of interest with two or more independently determinable classes of a solid-supported binding partner.
Assay techniques for determining the presence, desirability, and the concentration of an analyte using a binding partner having specificity for that analyte are frequently encountered, e.g., in the fields of biochemistry and clinical chemistry. Thus, for example, a wide range of immunological and related techniques has been proposed for determining materials, such as antigens in serum, using an appropriate binding partner for the analyte, such as a specific antibody, e.g., a monoclonal antibody, for a particular antigen.
One such technique comprises competitive binding assays, in which a known amount of a labeled version of an analyte to be determined, e.g., carrying a radioactive label, or its analogue and a relatively small known amount of a binding partner specific for the analyte are incubated with the analyte to be determined, whereby the labeled and the naturally occurring analyte compete for the binding partner. The amount of labeled analyte bound to the binding partner is inversely related to the concentration of analyte in the sample.
Another useful technique comprises sandwich assays. These employ an excess of the binding partner that binds to the analyte in the sample. A labeled second ligand is also added and binds to the captured analyte forming a sandwich, with the amount of label dependent on the amount of analyte captured. The amount of bound and labeled analyte is directly related to the concentration of analyte in the sample. The binding partner and the labeled ligand in such sandwich assays preferably have affinities for different binding sites, e.g., epitopes, on the analyte. The ligand may, for example, be labeled for reading on the basis of radioactivity, light absorption, or fluorescence.
Sandwich assays tend to exhibit greater sensitivity than competitive binding assays and are, therefore, usually preferred. It will be appreciated that high sensitivity is essential in, for example, immunoassays in clinical laboratories, where it maybe required to quantify, e.g., antigens present in the serum at concentrations in the nmol/l to pmol/l range or even lower. However, the sandwich assay requires the presence of two binding sites on the analyte. Therefore, the competitive format is preferred for smaller molecules where two binding sites may not be present or may be sterically hindered.
The binding partner in both of the above-described types of assays is commonly coupled to a solid support in order to facilitate isolation of the bound analyte and the competing or analyte-bound label. Thus, for example, the binding partner may be coupled to the surface of a reaction vessel, e.g., to the surfaces of the wells of a microtitre plate made from a suitable plastics material, so as to facilitate washing to remove unbound excess labeled ligand.
Alternatively, the binding partner may be coupled to the surfaces of an array of particles, for example, made of a suitable plastics material, such as polystyrene or polyacrylate. The separation of the bound analyte/label from the free label may then be affected by, for example, filtration or, in the event that superparamagnetic particles are employed, by the application of a magnetic field. The particles are advantageously of microscopic size in order to present a large total surface area coated with the binding partner. The use of monosized microparticles is preferred since it ensures that the particles exhibit standard binding properties.
An immunoassay for bead-based multiplexing uses coded particles with distinguishable optical characteristics (such as bead size, bead number, or incorporation of one or more fluorescent dyes). Particles with the same code receive the same ligand (e.g., antibody) on their surface and are thus responsive to the same analyte.
A disadvantage of the above-described basic assay techniques is that separation of the bound analyte and label and associated washing steps to remove the unbound label are inherently time-consuming and labor-intensive. It is known, however, that this problem may, in principle, be avoided in the case of particle-based assays if the particles are analyzed by means of flow cytometry. This typically involves the passage of a suspension of particles through the measurement region of a photometer in such a way that successive individual particles are irradiated with excitation light, causing the emission of a pulse of scattered light related to the size of the particle and a further signal, e.g., a pulse of fluorescent light, related to the amount and nature of the label bound to the particle. Accordingly, there is no need to separate unbound label prior to the flow cytometric particle analysis, which is therefore said to be a homogeneous, i.e., separation-free, assay.
A general problem associated with clinical assays especially immunoassays, using the above assay formats is that many analytes have wide clinical ranges. Thus, the signal response of such assays frequently saturates below the maximum clinical value. Specimens giving a saturated signal in such assays are typically diluted and rerun so that the assay range may cover more clinical values. Such a strategy is reasonable for single assays where the frequency of dilution and rerun is low. However, when a specimen is assayed for several analytes, the likelihood of needing to re-assay the specimen for a particular analyte increases. Since dilutions and reruns tie up system resources, an alternative method is needed for dealing with wide clinical range analytes in specimens.
U.S. Pat. No. 5,585,241 describes an assay technique that utilizes high and low affinity-binding partners, respectively, coated onto different types of monodisperse particles that are distinguishable by flow cytometry. Predetermined amounts of this binary particle mixture and of labeled ligand are incubated with the analyte, and the resulting two types of labeled ligand-carrying particles are thereafter independently, but simultaneously, detected by means of a flow cytometer, the analyte concentration being determined from the thus-obtained two measurement values by reference to a double standard calibration curve. However, this method requires the production or acquisition of two different types of binding partners (ligands) for the analyte. Further, each particle type requires a separate coupling reaction to attach the binding partner to the particle in addition to optimization of each of these coupling reactions.
U.S. Pat. No. 5,739,042 describes an assay in which two solid forms of binding partner are reacted successively, rather than simultaneously, with an analyte and a labeled ligand. The addition of the second form of the solid form of binding partner prevents any further binding of the analyte to the first form of solid binding partner and thus acts as a type of washing step in which the unbound analyte is xe2x80x9cwashed awayxe2x80x9d and thus effectively quenches the reaction. This method thus has the disadvantages of requiring an additional reagent delivery step. This step requires either associated hardware or a throughput time slice, both of which increases the cost required per unit of assay throughput.
There still exists a need for a cost-effective method of assaying one or more analytes in a sample without the need for sample dilution and rerunning the assay, and which provides for an extended dynamic range.
This invention relates to methods of assaying one or more analytes in a sample. More specifically, this invention provides assays that are capable of providing a wide dynamic range and rapid processing times, and eliminates the need for multiple sample dilutions and assay reruns.
Accordingly, one aspect of this invention provides a method for assaying an analyte in a sample, comprising:
a) providing a set of coated particles comprising at least
i) a first class of particles having a first binding partner for the analyte immobilized thereon, and
ii) a second class of particles having the first binding partner for the analyte immobilized thereon, wherein the first and second classes of particles are independently determinable, and wherein the particles of the first class are smaller than the particles of the second class;
b) simultaneously incubating the sample in the presence of the set of particles under conditions that allow the formation of a first complex comprising the sample analyte bound to the first binding partner immobilized on the first class of particles and/or a second complex comprising the sample analyte bound to the first binding partner immobilized on the second class of particles; and
c) determining the amount of the first complex and/or the second complex, wherein the amounts are determinative of the concentration of the analyte in the sample.
In one embodiment, the assay is a sandwich assay, wherein the method further comprises incubating the sample and the set of coated particles with a ligand comprising a labeled second binding partner for the analyte.
In another embodiment, the assay is a competitive assay, wherein the method further comprises incubating the sample and the set of coated particles with a ligand comprising a labeled analogue of the analyte.
The methods of this invention can be used to assay a single analyte or multiple analytes simultaneously.
Additional advantages and novel features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The novel features and advantages of the invention may be realized and attained by means of the instrumentalities, combinations, and methods particularly pointed out in the appended claims.